Infectious diarrhea is a complex syndrome caused by many viruses, parasites, fungi and bacteria such as Salmonella. Salmonellosis itself encompasses a spectrum of clinical diseases that range from enteric fever, a serious condition that kills about 600,000 people a year, to non- typhoidal zoonotic infections that affect more than a million Americans annually. Treatment of Salmonella and medically important Gram-negative rods is often complicated by the increasing resistance of pathogenic bacteria to antibiotics used in the clinic. The parasitic relationship between Salmonella and host mononuclear phagocytes is one of the hallmarks of salmonellosis. The survival of Salmonella within macrophages relies on the type III secretion system encoded within the Salmonella pathogenicity island 2 (SPI2). The expression of SPI2 is controlled by the SsrA/SsrB two-component regulatory system. In addition to being regulated by canonical phosphorylation of a conserved aspartate in the receiver domain, our investigations indicate that SsrB is under the post-translational control via the oxidation and S-nitrosylation of Cys203 in the dimerization domain. Chaperone and disulfide reductase activities of TrxA further regulate SsrB function, thereby boosting SPI2-dependent antioxidant defenses. We hypothesize that the SPI2 master regulator SsrB is under post-translational control of oxidative modifications of the Cys203 thiol group in the dimerization domain, and protein-protein interactions with the disulfide reductase and chaperone functions of thioredoxin 1. Aim 1 will determine the molecular mechanisms by which SsrB redox and oxidation states regulate SPI2 transcription. Aim 2 will explore the role that specific charged and hydrophobic residues play in the ability of redox active SsrB Cys203 to sense oxidative and nitrosative stress. Aim 3 will characterize the mechanisms by which the chaperone and disulfide reductase activities of thioredoxin 1 regulate SsrB function and Salmonella pathogenesis. These investigations will provide profound insights into the antioxidant and antinitrosative defenses of Salmonella that are critical for intracellular survival and virulence. Specifically, the proposed research will elucidate the previously unappreciated role for reactive species and an antioxidant protein in the post-translational regulation of SsrB, the intracellular survival of Salmonella, and the pathogenesis of this intracellular bacterium in a murine model of salmonellosis.